US4751190A - Fluorescence polarization immunoassay and reagents for use therein - Google Patents
Fluorescence polarization immunoassay and reagents for use therein Download PDFInfo
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- US4751190A US4751190A US06/847,801 US84780186A US4751190A US 4751190 A US4751190 A US 4751190A US 84780186 A US84780186 A US 84780186A US 4751190 A US4751190 A US 4751190A
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- tracer
- ligand
- crp
- antibody
- fluorescence polarization
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- NRHMKIHPTBHXPF-TUJRSCDTSA-M sodium cholate Chemical compound [Na+].C([C@H]1C[C@H]2O)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC([O-])=O)C)[C@@]2(C)[C@@H](O)C1 NRHMKIHPTBHXPF-TUJRSCDTSA-M 0.000 description 1
- 239000012064 sodium phosphate buffer Substances 0.000 description 1
- 229930002534 steroid glycoside Natural products 0.000 description 1
- 150000008143 steroidal glycosides Chemical class 0.000 description 1
- 101150035983 str1 gene Proteins 0.000 description 1
- 229960005322 streptomycin Drugs 0.000 description 1
- 239000007929 subcutaneous injection Substances 0.000 description 1
- 238000010254 subcutaneous injection Methods 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 229960000278 theophylline Drugs 0.000 description 1
- 210000001685 thyroid gland Anatomy 0.000 description 1
- 229940034208 thyroxine Drugs 0.000 description 1
- XUIIKFGFIJCVMT-UHFFFAOYSA-N thyroxine-binding globulin Natural products IC1=CC(CC([NH3+])C([O-])=O)=CC(I)=C1OC1=CC(I)=C(O)C(I)=C1 XUIIKFGFIJCVMT-UHFFFAOYSA-N 0.000 description 1
- 229960000707 tobramycin Drugs 0.000 description 1
- NLVFBUXFDBBNBW-PBSUHMDJSA-N tobramycin Chemical compound N[C@@H]1C[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N NLVFBUXFDBBNBW-PBSUHMDJSA-N 0.000 description 1
- 210000002700 urine Anatomy 0.000 description 1
- MSRILKIQRXUYCT-UHFFFAOYSA-M valproate semisodium Chemical compound [Na+].CCCC(C(O)=O)CCC.CCCC(C([O-])=O)CCC MSRILKIQRXUYCT-UHFFFAOYSA-M 0.000 description 1
- 229960000604 valproic acid Drugs 0.000 description 1
- 229960003165 vancomycin Drugs 0.000 description 1
- MYPYJXKWCTUITO-UHFFFAOYSA-N vancomycin Natural products O1C(C(=C2)Cl)=CC=C2C(O)C(C(NC(C2=CC(O)=CC(O)=C2C=2C(O)=CC=C3C=2)C(O)=O)=O)NC(=O)C3NC(=O)C2NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(CC(C)C)NC)C(O)C(C=C3Cl)=CC=C3OC3=CC2=CC1=C3OC1OC(CO)C(O)C(O)C1OC1CC(C)(N)C(O)C(C)O1 MYPYJXKWCTUITO-UHFFFAOYSA-N 0.000 description 1
- MYPYJXKWCTUITO-LYRMYLQWSA-O vancomycin(1+) Chemical compound O([C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1OC1=C2C=C3C=C1OC1=CC=C(C=C1Cl)[C@@H](O)[C@H](C(N[C@@H](CC(N)=O)C(=O)N[C@H]3C(=O)N[C@H]1C(=O)N[C@H](C(N[C@@H](C3=CC(O)=CC(O)=C3C=3C(O)=CC=C1C=3)C([O-])=O)=O)[C@H](O)C1=CC=C(C(=C1)Cl)O2)=O)NC(=O)[C@@H](CC(C)C)[NH2+]C)[C@H]1C[C@](C)([NH3+])[C@H](O)[C@H](C)O1 MYPYJXKWCTUITO-LYRMYLQWSA-O 0.000 description 1
- 239000011800 void material Substances 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/533—Production of labelled immunochemicals with fluorescent label
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
- G01N33/542—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with steric inhibition or signal modification, e.g. fluorescent quenching
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/72—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood pigments, e.g. haemoglobin, bilirubin or other porphyrins; involving occult blood
- G01N33/728—Bilirubin; including biliverdin
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S436/00—Chemistry: analytical and immunological testing
- Y10S436/80—Fluorescent dyes, e.g. rhodamine
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S436/00—Chemistry: analytical and immunological testing
- Y10S436/805—Optical property
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S436/00—Chemistry: analytical and immunological testing
- Y10S436/825—Pretreatment for removal of interfering factors from sample
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S436/00—Chemistry: analytical and immunological testing
- Y10S436/826—Additives, e.g. buffers, diluents, preservatives
Definitions
- the present invention relates generally to a method, and reagents useful in the method, for determining ligands in liquids, especially biological fluids such as serum, plasma, spinal fluid, amnionic fluid and urine.
- the present invention relates more particularly to novel fluorescence polarization immunoassays which employ dioctyl sodium sulfosuccinate as a surfactant to improve the performance of the assay.
- ligand binding immunoassays for measuring ligands are well known, and are based on the competition between a ligand in a test sample and a labeled reagent, referred to as a tracer, for a limited number of receptor binding sites on antibodies specific to the ligand and tracer.
- concentration of ligand in the sample determines the amount of tracer that will specifically bind to an antibody.
- the amount of tracer-antibody complex produced can be quantitatively measured and is inversely proportional to the quantity of ligand in the test sample.
- Fluorescence polarization immunoassay techniques are based on the principle that a fluorescent labeled compound when excited by plane polarized light, will emit fluorescence having a degree of polarization inversely related to its rate of rotation. Specifically, when a molecule such as a tracer-antibody complex having a fluorescent label is excited with plane polarized light, the emitted light remains highly polarized because the fluorophore is constrained from rotating between the time when light is absorbed and when it is emitted.
- the analyte may be substantially larger, e.g., having a molicular weight on the order of 100,000 daltons or more, as long as it is capable of measurement using fluorescent polarization immunoassay techniques (see, for example, co-pending U.S. application Ser. No. 757,822 filed July 22, 1985, the disclosure of which is incorporated herein by reference, which describes a fluorescent polarization immunoassay for C-reactive protein having a molecular weight of about 120,000 daltons).
- Antibody specific to the analyte and the tracer is also included in the mixture. The tracer and the ligand compete for a limited number of receptor binding sites on the antibody. The amount of tracer that will bind is inversely related to the concentration of analyte in the sample, since the analyte and tracer each bind to the antibody in proportion to their respective concentrations.
- the TDx@ Fluorescence Polarization Analyzer an instrument commercially available from Abbott Laboratories, Abbott Park, Ill., is an automated system for the performance, inter alia, of fluorescence polarization assays.
- the TDx@ Analyzer has achieved remarkable commercial success in providing fluorescent polarization immunoassays to clinical laboratories for the determination in patient samples of many ligands, including antiasthmatic drugs, such as theophylline, antiarrhythmic drugs, such as lidocaine, N-acetylprocainamide, procainamide and quinidine, antibiotic drugs, such as amikacin, gentamicin, kanamycin, netilmicin, streptomycin, tobramycin and vancomycin, anticonvulsant drugs, such as carbamazepine, phenytoin, phenobarbital, primidone, and valproic acid, antineoplastic drugs, such as methotrexate, cardiac glycosides, such as digoxin, thyroid function assays
- icteric serum or plasma can contribute a significant error to the desired polarization measurement.
- a major fluorescent component of icteric serum or plasma is albumin-bound bilirubin. Bilirubin is the final product of heme catabolism and in normal individuals is present in serum at less than 1 mg/dl. In various disease states affecting the liver, bilirubin is markedly elevated, reaching 10-20 mg/dl in some cases. Neonates ofter attain high levels in the 10-20 mg/dl range due to poor liver function immediately post-partum.
- 4,492,762 discloses conducting fluorescent polarization immunoassays in a solution containing effective amounts of an anionic surfactant to disrupt bilirubin-serum albumin complex in the sample and thereby reduce background fluorescence of the sample.
- the '762 patent discloses that a broad category of anionic surfactants are useful for this purpose and that concentration ranges of 0.001 to 0.2 (weight/volume) percent are preferred.
- Preferred surfactants for use in the practice of the method of the '762 patent have been sodium dodecyl sulfate and sodium cholate.
- dioctyl sodium sulfonate is employed as an anionic surfactant in fluorescence polarization immunoassays to reduce background resulting from bilirubin-serum albumin complex in serum samples. It has been unexpectantly found that dioctyl sodium sulfonate is highly effective for this purpose at relatively low concentrations with little or no degredation effect on antibody employed in the assay, thereby permitting the use of fluorescence polarization techniques to determine the concentration of analytes heretofore precluded by prior art methods.
- the invention further encompasses certain novel reagents useful in the above-described method.
- DSS or dioctyl sodium sulfonate as used herein is the compound sulfobutanedioic acid 1,4-bis(2-ethylhexyl)ester sodium salt having the structural formula: ##STR1##
- other salts such as the potassium salt, calcium salt, lithium salt, magnesium salt and other equivalent salts, are included within the scope of the invention as set forth herein.
- ligand refers to a molecule such as a hapten, to which a binding protein, such as a receptor or an antibody, can be obtained or formed.
- haptens are protein-free compounds, generally of low molecular weight, which do not induce antibody formation when injected into an animal, but which are reactive to antibodies.
- ligand may refer to a moderate to high molecular weight compounds such as proteins, e.g., C-reactive protein.
- Antibodies to haptens are generally raised by first conjugating the haptens to a protein and injecting the conjugate product into an animal. The resulting antibodies are isolated by conventional, well-known antibody isolation techniques.
- ligand-analog refers to a mono- or polyvalent radical, a substantial portion of which has the same spatial and polar organization as the ligand to define one or more determinant or epitopic sites capable of competing with the ligand for the binding sites of a receptor.
- a characteristic of such ligand-analog is that it possesses sufficient structural similarity to the ligand of interest so as to be recognized by the antibody for the ligand.
- the ligand-analog will have the same or substantially the same structure and charge distribution (spatial and polar organization) as the ligand of interest (for purposes of the present invention, CRP) for a significant portion of the molecular surface.
- the linking site for a hapten will be the same in preparing the antigen for production of antibodies as that used in the tracer for linking to the ligand, the same portion of the ligand analog which provides the template for the antibody will be exposed by the ligand analog in the tracer.
- the present invention involves the use of fluorescein and derivatives of fluorescein.
- a necessary property of fluorescein and its derivatives for the usefulness of the tracer compounds is the fluorescence of fluorescein. These compounds provide the fluorescent response when excited by polarized light of an appropriate wavelength, thereby to enable the fluorescence polarization measurement to be made.
- the tracer compounds used in the assay provided by the present invention are formed of conjugates of the ligand to be determined, or a ligand-analog, with fluorescein or a fluorescein derivative, and exist in solution as biologically acceptable salts such as sodium, potassium, ammonium and the like, which allows the compounds to exist in the open, fluorescent form, when employed in the analytical methods of the present invention.
- the specific salt present depends on the buffer employed to adjust the pH level, for example, in the presence of a sodium phosphate buffer, the compounds utilized in the present invention will generally exist in the open form, as a sodium salt.
- Suitable fluorescein tracer compounds for use in the invention include, for example, carboxyfluorescence fluorescein isothiocynates (FITC), triazinylaminofluoresceins (DTAF) and many other compounds well kown in the art, including those disclosed in the art previously cited.
- FITC carboxyfluorescence fluorescein isothiocynates
- DTAF triazinylaminofluoresceins
- the selection of a particular fluoerescent tracer for use is a matter of choice for the routine, given the teachings hereof, and is not crucial to the practice of the present invention.
- a sample containing a ligand to be determined is intermixed with a tracer and an antibody specific for the ligand and the tracer in the presence of DSS.
- Concentration ranges of DSS in the reaction mixture effective in the practice of the invention will depend on the specific ligand, antibody and other reagents employed in the assay, but will generally be in the range of about 0.001 to about 1.0 percent (weight/volume), more preferably about 0.002 to about 0.5 percent (weight/volume) and most preferably about 0.005 to about 0.1 percent (weight/volume).
- the ligand present in the sample and the tracer compete for a limited number of antibody sites, resulting in the formation of ligand-antibody and tracer-antibody complexes.
- the ratio of ligand-antibody complex to tracer-antibody complex that is formed is directly proportional to the amount of ligand present in the sample. Therefore, upon exciting the mixture with polarized light and measuring the polarization of the fluorescence emitted by a tracer and a tracer-antibody complex, one is able quantitatively to determine the amount of ligand in the sample.
- a tracer in solution which is not complexed to an antibody is free to rotate in less than the time required for absorption, and re-emitted light is relatively randomly oriented so that the fluorescence polarization of a tracer not complexed to an antibody is low, approaching zero.
- the tracer-antibody complex thus formed assumes the rotation of the antibody molecule which is slower than that of the relatively small tracer molecule, thereby increasing the polarization observed. Therefore, when a ligand competes with the tracer for antibody sites, the observed polarization of fluorescence of the resulting mixture of the free tracer and tracer-antibody complex assumes a value intermediate between that of the tracer and that of the tracer-antibody complex.
- the observed polarization value is closer to that of the free ligand, i.e., low. If the test sample contains a low concentration of the ligand, the polarization value is closer to that of the bound ligand, i.e., high.
- the pH at which the method of the present invention is practiced must be sufficient to allow the tracers to exist in their ionized state.
- the pH maY range from about 3 to 12, more usually in the range of from about 5 to 10, most preferably from about 6 to 9.
- Various buffers can be used to achieve and maintain the pH during the assay procedure. Representative buffers include borate, acetate, phosphate, carbonate, tris, barbital and the like.
- the particular buffer employed is not critical to the present invention, but in an individual assay, a specific buffer may be preferred in view of the antibody employed and ligand to be determined.
- the cation portion of the buffer will generally determine the cation portion of the tracer salt in solution.
- the methods of the present invention are practiced at moderate temperatures and preferably at a constant temperature.
- the temperature will normally range from about zero degrees to about 50 degrees C., more usually from about 15 degrees to about 40 degrees C.
- concentration of ligand which can be assayed in accordance with the invention will generally vary from about 10 -2 to about 10 -13 M, more usually from about 10 -4 to about 10 -10 M. High concentrations of ligand can be assayed upon dilution of the original sample.
- concentration range of ligand In addition to the concentration range of ligand, considerations such as whether the assay is qualitative, semiquantitative or quantitative, the equipment employed, and the characteristics of the tracer and antibody will normally determine the concentration of the tracer and antibody which is used. While the concentration range of ligand in the sample will determine the range of concentration of the other reagents, i.e., tracer and antibody, normally to optimize the sensitivity of the assay, individual reagent concentrations will be determined empirically. Appropriate concentrations of the tracer and antibody are readily ascertained by one of ordinary skill in the art.
- the fluorescence polarization immunoassay for ligands provided by the present invention can be performed especially advantageously using reagents and assay procedures, in accordance with the invention, on a TDx (registered trademark) Fluorescence Polarization Analyzer, commercially available from Abbott Laboratories, Abbott Park, Ill., from whom full details concerning operation and features of this Analyzer are available.
- TDx registered trademark Fluorescence Polarization Analyzer
- the present invention also contemplates a reagent for use in the assays of the invention, which, upon dilution in the performance of the assay, will result in a DSS concentration in the reaction mixture within the concentration ranges heretofore described.
- concentration of DSS in the reagent will vary depending on the assay protocol for which it is intended to be used, but will advantageously comprise from about 0.01 to about 20.0 percent (weight/volume) DSS, more preferably about 0.02 to about 15.0 percent (weight/volume) DSS and most preferably about 0.05 to about 10.0 percent (weight/volume) DSS.
- the solvent system of the reagent of the invention may be an aqueous solvent or an organic solvent which is miscible in water, such as one or more of ethylene glycol, propylene glycol, DMSO, DMF, lower alkanols or the like.
- One presently particularly preferred solvent system is a mixture of 2-propanol, DMSO and propylene glycol.
- the reagent comprising DSS will preferably be a pretreatment buffer solution for use in the assay.
- a measured volume of standard or test serum is delivered into a test tube and diluted with a pretreatment buffer comprising DSS;
- the amount of tracer bound to antibody is measured by fluorescence polarization techniques as a measure of the amount of ligand in the sample.
- CRP is obtained from malignant ascitic and pleural fluids by calcium-dependent affinity chromatography on pneumococcal C-polysaccharide covalently coupled by cyanogen bromide-activated Sepharose. It is then gel filtered on Ultrogel AcA44 (acrylamide-agarose beads) in the presence of calcium ions, combining molecular sieve chromatography with removal of contaminating SAP by its affinity for agarose. Residual trace contaminants are removed by immunoabsorption with anti-normal human serum and anti-SAP antibodies insolubilised on Sepharose and by absorption with Sepharose-Con A to remove glycoproteins and Blue-Sepharose to remove albumin. After a final gel filtration step on Sephacryl S-300, between 35-40% of the initial CRP is recovered in substantially pure form.
- Sheep and goats are immunized by deep subcutaneous or intramuscular injections of isolated CRP emulsified in complete Freund's adjuvant, followed by biweekly or monthly booster injections of emulsions in incomplete adjuvant. Each injection contains at least 500 microgramns CRP. This regiment is followed for all animals for a five month period while the animals are monitored for antibody titer at biweekly or monthly bleeding intervals. Booster injections are then interrupted for three months while titer monitoring continued. Following the three months rest, boosting is resumed as titers begin to drop.
- a stock solution of 5-(4,6-dichloro-triazin-2-yl)-Amino Fluorescein, (DTAF) is prepared in absolute ethanol with the aid of sonification, at 2 mg/ml.
- Stock CRP contains in 0.005 Molar Borate buffer, pH 9.0, 0.002 Molar CaCL 2 and 0.9% NaCl at 3 mg/ml, is made to a concentration of 500 micrograms in 0.04 Molar Borate buffer pH 9.0, 0.002M CaCl 2 , 0.9% NaCl.
- To 1 ml of the (500 micrograms/ml) CRP solution 25 microliters of stock DTAF is added. The coupling reaction is then carried out for 1 hour at ambient temperature with mixing, in the dark.
- the DTAF reaction is quenched with 50 ul of 10% glycine prepared in 0.04 Molar borate buffer, pH 9.0 (same buffer as above), and incubated with mixing for 15 minutes at the above conditions.
- the conjugate is chromatographed over sephodex G-25 and eluted with Borate buffer pH 9.0 (same buffer as above) and collected at void volume. It is then diluted to a desired concentration for use in an automated assay on the TDx Analyzer.
- the stock DTAF/CRP conjugate is diluted in buffer containing protein and salt stabilizers and 0.1% NaN 3 as preservative, to give a net intensity reading of 3000 at gain of 20 on the TDx Analyzer.
- Antiserum Dilutions of raw CRP antiserum are made at from 1:10 to 1:100. 25 ul of each dilution is added to a cuvette and allowed to incubate with 25 ul of tracer reagent in 2 ml final volume of 0.1M Phosphate buffer, pH 7.5, 0.01% Bovine-gamma ethylene glyco-Globulin (BGG), 0.1% NaN 3 and 2% (by Volume). The antiserum and tracer react for 3.4 minutes at 35° C. in the above buffered conditions. A dilution factor is determined for the antiserum which is based on the fluorescence polarization measured. The antiserum reagent is then prepared in the above phosphate buffer by diluting the raw antiserum according to the determined dilution factor.
- BGG Bovine-gamma ethylene glyco-Globulin
- Pretreatment buffer reagent consists of a solution of 3.5% (weight/volume) DSS in 0.05 Molar Tris, pH 8.0, 0.1% NaN 3 as preservative and other organic stabilizing solvents.
- TDx assay buffer consists of 0.1 Molar Phosphate, pH 7.5, 0.01% Bovine-gamms-Globulin (BGG) and 0.1% NaN 3 as a preservative.
- Calibrators C-Reactive protein is placed in buffered synthetic serum matrix containing protein and salt stabilizers and 0.1% NaN 3 as a preservative.
- the pH is adjusted to 8.0 with 6N HCl.
- This pretreatment composition has been found to be effective in eliminating Bilirubin interference at Bilirubin concentrations of 20 mg/dl while using a CRP assay sample volume of 8.0 microliters.
- a fluorescence polarization immunoassay (FPIA) for CRP is carried out on the TDx Fluorescense Polarization Analyzer as follows.
- the reaction sequence, incubation, timing, reagent volumes and sample volumes are microprocessor controlled according to programmed assay parameters.
- specimens and reagents are loaded on the TDx Analyzer, in their respective receptacles.
- Specimen, antiserum, pretreatment reagent and buffer are dispensed into the reaction well.
- One-half of the final volume of the diluted specimen is dispensed into the cuvette along with sufficient buffer to give one-half the final reaction volume.
- a background intensity reading is taken on the mixture of specimen, antiserum and pretreatment reagent.
- the second half of the diluted specimen is dispensed into the cuvette with tracer and buffer to provide the final reaction volume of 2 ml.
- the final intensity measurement is then made.
- Steps 1-4 are repeated for each sample and are accomplished in 18.4 seconds per sample.
- the cuvette contents are incubated for 6.4 minutes at 34° C. while a background reading is taken at 3.4 minutes and stored for each specimen.
- the final cuvette reaction mixture is incubated for 3.4 minutes and a final reading taken for each specimen.
- the net polarization reading is converted to a CRP concentration by utilizing four stored mathematical constants derived from a calibration curve, previously generated with calibrators of known CRP concentration.
- the four constants are determined by a least-square curve-fit four parameter program which is part of the date-handling system associated with the TDx Analzyer.
- a stock solution of bilirubin (Sigma Chemical Co., Cat. No. B40126) is prepared at 1000 mg/dl in 0.1M NaOH.
- the stock solution is used to spike low (L), medium (M) and high (H) CRP-containing standard serum samples to obtain bilirubin concentrations in the samples as set forth below.
- the samples are then assayed according to the foregoing procedure using (a) the pretreatment reagent composition comprising DSS previously described, or (b) the pretreatment reagent composition previously described but comprising 0.7% sodium dodecyl sulfate (SDS) and 0.5% lauryl dodecyl sulfate (LDS) in place of DSS.
- SDS sodium dodecyl sulfate
- LDS lauryl dodecyl sulfate
- Human serum specimens are obtained for a period of one and one-half months from a patient population requested for CRP testing.
- a CRP value for each specimen is generated at the hospital utilizing a commercial nephelometric method. Samples are transported frozen, then tested by the CRP assay as aforedescribed. Patient results from both methods are compared by linear regression analysis. The following results are indicated for the 345 specimens tested:
- NPM Nephelomethy
- RID radialimmunodiffusion
- TDx CRP TDx CRP
- a detection limit of 0.3 mg/dl was based on two standard derivatives taken away from the millipolarization (mP) mean of twenty "zero" calibrator replicates. The resulting mP was then read of the calibration curve found to correspond to a CRP concentration of 0.3 mg/dl.
Abstract
Description
______________________________________ Concentration Constituent grams/liter Other ______________________________________ Tris 12.11 (0.1 Molar) Na.sub.2 SO.sub.4 20.0 (2%) (Anhydrous) Ovalbumin 5.0 (0.5%) Hydrolysate Propylene Glycol 20.0 ml (2% by volume) CaCl.sub.2 2H.sub.2 O 0.294 (0.002 Molar) NaN.sub.3 1.0 (0.1%) ______________________________________ Adjust pH with: 6 N HCl to 7.0
______________________________________ Concentration Constituent grams/liter Other ______________________________________ Tris 12.11 (0.1 Molar) Na.sub.2 SO.sub.4 8.0 (8.0%) Ovalbumin 10.0 (1.0%) Hydrolysate CaCl.sub.2 2H.sub.2 O 0.294 (0.002 Molar) NaN.sub.3 1.0 (0.1%) ______________________________________ The pH is adjusted with 6 N HCl to pH 8.0.
______________________________________ grams or milliliter/ Constituent Concentration liter ______________________________________ Tris 0.05 Molar 6.06 g NaN.sub.3 0.1% 1.00 g 2-Propanol 10% by Volume 100.0 ml DMSO 25% by Volume 200.0 ml Propylene Glycol 5% by Volume 20.0 ml Dioctyl Sodium 4% 66.67 ml Sulfosuccinate at 60% Stock Concentration ______________________________________
______________________________________ Initial Concentrated Measured Sample CRP CRP Added CRP Conc. % Conc. mg/dl mg/dl mg/dl Recovery ______________________________________ .15 2.19 2.34 100 .15 10.96 11.11 100 .15 21.92 21.99 99.6 1.73 9.55 11.17 98.9 1.68 9.55 10.98 97.4 ______________________________________
______________________________________ Bilirubin Conc. CRP SDS/LDS % DSS % mg/dl Level Tested Interference Interference ______________________________________ 4.8 L 17.1 4.8 M 4.3 -0.8 H 8.3 0.9 9.8 L 30.9 -4.9 M 5.6 -2.1 H 11.1 3.5 14.8 L 41.5 2.8 M 7.2 1.9 H 12.2 1.9 20.3 L 100+ 6.1 M 9.6 -0.4 H 18.8 4.1 24.6 L 100+ 18.1 M 7.3 4.8 H 18.0 7.9 ______________________________________
______________________________________ Method Compared Slope Intercept r ______________________________________ TDx CRP vs. NPM CRP 1.06 -0.17 0.99 TDx CRP vs. RID CRP 0.97 0.40 0.99 NPM CRP vs. RID CRP 0.91 0.50 0.98 ______________________________________
Claims (3)
Priority Applications (5)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US06/847,801 US4751190A (en) | 1985-07-22 | 1986-04-03 | Fluorescence polarization immunoassay and reagents for use therein |
EP87104890A EP0240021B1 (en) | 1986-04-03 | 1987-04-02 | Fluorescence polarization immunoassay and reagents for use therein |
CA000533669A CA1287798C (en) | 1986-04-03 | 1987-04-02 | Fluorescence polarization immunoassay and reagents for use therein |
DE8787104890T DE3774739D1 (en) | 1986-04-03 | 1987-04-02 | FLUORESCENCE POLARIZATION IMMUNOASSAYS AND REAGENTS TO BE USED IN THEM. |
JP62081347A JPH0785086B2 (en) | 1986-04-03 | 1987-04-03 | Fluorescence polarization immunoassay and reagents used in the assay |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US75782285A | 1985-07-22 | 1985-07-22 | |
US06/847,801 US4751190A (en) | 1985-07-22 | 1986-04-03 | Fluorescence polarization immunoassay and reagents for use therein |
Related Parent Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US75782285A Continuation-In-Part | 1985-07-22 | 1985-07-22 |
Publications (1)
Publication Number | Publication Date |
---|---|
US4751190A true US4751190A (en) | 1988-06-14 |
Family
ID=25301549
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US06/847,801 Expired - Fee Related US4751190A (en) | 1985-07-22 | 1986-04-03 | Fluorescence polarization immunoassay and reagents for use therein |
Country Status (5)
Country | Link |
---|---|
US (1) | US4751190A (en) |
EP (1) | EP0240021B1 (en) |
JP (1) | JPH0785086B2 (en) |
CA (1) | CA1287798C (en) |
DE (1) | DE3774739D1 (en) |
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WO2022034467A1 (en) | 2020-08-10 | 2022-02-17 | Mesoblast International Sarl | A composition comprising mesenchymal precursor or stem cells and their use |
WO2024009226A1 (en) | 2022-07-05 | 2024-01-11 | Mesoblast International Sarl | Cryopreserved intermediate and potency assay for same |
Also Published As
Publication number | Publication date |
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DE3774739D1 (en) | 1992-01-09 |
JPS62239057A (en) | 1987-10-19 |
EP0240021B1 (en) | 1991-11-27 |
EP0240021A1 (en) | 1987-10-07 |
JPH0785086B2 (en) | 1995-09-13 |
CA1287798C (en) | 1991-08-20 |
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